Identification of stathmin as a novel marker of cell proliferation in the recovery phase of acute ischemic renal failure

Ischemic renal injury can be classified into the initiation and extension phase followed by the recovery phase. The recovery phase is characterized by increased dedifferentiated and mitotic cells in the damaged tubules. Suppression subtractive hybridization was performed by using RNA from normal and ischemic kidneys to identify the genes involved in the physiological response to ischemia-reperfusion injury (IRI). The expression of stathmin mRNA increased by fourfold at 24 h of reperfusion. The stathmin mRNA did not increase in sodium-depleted animals or in animals with active, persistent injury secondary to cis-platinum. Immunofluorescent labeling demonstrated that the expression of stathmin increased dramatically at 48 h of reperfusion. Labeling with antibodies to stathmin and proliferating cell nuclear antigen (PCNA) indicates that the expression of stathmin was induced before the upregulation of PCNA and that all PCNA-positive cells expressed stathmin. Double immunofluorescent labeling demonstrated the colocalization of stathmin with vimentin, a marker of dedifferentiated cells. Stathmin expression was also significantly enhanced in acute tubular necrosis in humans. On the basis of its induction profile in IRI, the data indicating its enhanced expression in proliferating cells and regenerating organs, we propose that stathmin is a marker of dedifferentiated, mitotically active epithelial cells that may contribute to tubular regeneration and could prove useful in distinguishing the injury phase from recovery phase in IRI.     

Incorporation of SPION-casein core-shells into silk-fibroin nanofibers for cardiac tissue engineering

Mimicking the structure of extracellular matrix (ECM) of myocardium is necessary for fabrication of functional cardiac tissue. The superparamagnetic iron oxide nanoparticles (SPIONs, Fe₃O₄), as new generation of magnetic nanoparticles (NPs), are highly intended in biomedical studies. Here, SPION NPs (1 wt%) were synthesized and incorporated into silk-fibroin (SF) electrospun nanofibers to enhance mechanical properties and topography of the scaffolds. Then, the mouse embryonic cardiac cells (ECCs) were seeded on the scaffolds for in vitro studies. The SPION NPs were studied by scanning electron microscope (SEM), X-ray diffraction (XRD), and transmission electron microscope (TEM). SF nanofibers were characterized after incorporation of SPIONs by SEM, TEM, water contact angle measurement, and tensile test. Furthermore, cytocompatibility of scaffolds was confirmed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. SEM images showed that ECCs attached to the scaffolds with elongated morphologies. Also, the real-time PCR and immunostaining studies approved upregulation of cardiac functional genes in ECCs seeded on the SF/SPION-casein scaffolds including GATA-4, cardiac troponin T, Nkx 2.5, and alpha-myosin heavy chain, compared with the ones in SF. In conclusion, incorporation of core-shells in SF supports cardiac differentiation, while has no negative impact on ECCs' proliferation and self-renewal capacity.     

Effect of feeding and sludge age on acclimated bacterial community and fate of slowly biodegradable substrate

The study investigated the effect of feeding regime and sludge age on starch utilization. For this purpose, parallel sequencing batch reactors were operated with pulse and continuous feeding of soluble starch at sludge ages of 8 and 2 days. Pulse feeding induced almost complete conversion of starch to glycogen, while storage was lowered and accompanied with direct growth under continuous feeding, regardless of sludge age. Low sludge age did not alter simultaneous storage and utilization for direct growth but it slightly favoured direct utilization due to faster growing biomass. Experimental results suggested adsorption of starch onto biomass as a preliminary removal mechanism prior to hydrolysis at sludge age of 8 days. Adsorption was not noticeable as substrate removal, glycogen generation and dissolved oxygen decrease were synchronous at sludge age of 2 days. Bacterial community always included fractions storing glycogen although sludge age only affected the relative magnitude of filamentous growth.     

Enhanced cadmium-induced testicular necrosis and renal proximal tubule damage caused by gene-dose increase in a Slc39a8-transgenic mouse line

Resistance to cadmium (Cd)-induced testicular necrosis is an autosomal recessive trait defined as the Cdm locus. Using positional cloning, we previously identified the Slc39a8 (encoding an apical-surface ZIP8 transporter protein) as the gene most likely responsible for the phenotype. In situ hybridization revealed that endothelial cells of the testis vasculature express high ZIP8 levels in two sensitive inbred mouse strains and negligible amounts in two resistant strains. In the present study, we isolated a 168.7-kb bacterial artificial chromosome (BAC), carrying only the Slc39a8 gene, from a Cd-sensitive 129/SvJ BAC library and generated BAC-transgenic mice. The BTZIP8-3 line, having three copies of the 129/SvJ Slc39a8 gene inserted into the Cd-resistant C57BL/6J genome (having its normal two copies of the Slc39a8 gene), showed tissue-specific ZIP8 mRNA expression similar to wild-type mice, mainly in lung, testis, and kidney. The 2.5-fold greater expression paralleled the fact that the BTZIP8-3 line has five copies, whereas wild-type mice have two copies, of the Slc39a8 gene. The ZIP8 mRNA and protein localized especially to endothelial cells of the testis vasculature in BTZIP8-3 mice. Cd treatment reversed Cd resistance (seen in nontransgenic littermates) to Cd sensitivity in BTZIP8-3 mice; reversal of the testicular necrosis phenotype confirms that Slc39a8 is unequivocally the Cdm locus. ZIP8 also localized specifically to the apical surface of proximal tubule cells in the BTZIP8-3 kidney. Cd treatment caused acute renal failure and signs of proximal tubular damage in the BTZIP8-3 but not nontransgenic littermates. BTZIP8-3 mice should be a useful model for studying Cd-induced disease in kidney. kidney; testis; ZIP8; bacterial artificial chromosome     

Experimentation on Degradation of Petroleum in Contaminated Soils in the Root Zone of Maize (Zea Mays L.) Inoculated with Piriformospora Indica

Plant-based methods such as rhizodegradation are very promising for the remediation of petroleum-contaminated soils. Associations of plants with endophytes can further enhance their phytoremediation potential. In this study, a rhizobox experiment was conducted to investigate whether inoculation with the root-colonizing fungus Piriformospora indica could further enhance the degradation of petroleum hydrocarbons in the root zone of maize (Zea mays L.). The rhizoboxes were subdivided into compartments in accordance with distance from the plants. After filling the boxes with soil from a petroleum-contaminated site, seedlings that had either been inoculated with P. indica or not were grown in the middle compartments of the rhizoboxes and grown for 64 days. A plant-free treatment was included for control. The presence of roots strongly increased the counts of total and petroleum-degrading soil bacteria, respiration, dehydrogenase activity, water-soluble phenols and petroleum degradation. All these effects were also found in the soil adjacent to the middle compartments of the rhizoboxes, but strongly decreased further away from it. Inoculation with P. indica further enhanced all the recorded parameters without changing the spatial pattern of the effects. Inoculated plants also produced around 40% more root and shoot biomass than noninoculated plants and had greener leaves. Together, the results indicate that the treatment effects on the recorded soil microbial and biochemical parameters including petroleum hydrocarbon degradation were primarily due to increased root exudation. Irrespectively of this, they show that maize can be used to accelerate the rhizodegradation of petroleum hydrocarbons in soil and that inoculation with P. indica can substantially enhance the phytoremediation performance of maize.     

Biological behavior of the curcumin incorporated chitosan/poly(vinyl alcohol) nanofibers for biomedical applications

Electrospun composite scaffolds show high ability to be used in regenerative medicine and drug delivery, due to the nanofibrous structure and high surface area to volume ratio. In this study, we used nanofibrous scaffolds fabricated by chitosan (CS), poly(vinyl alcohol) (PVA), carbopol, and polycaprolactone using a dual electrospinning technique while curcumin (Cur) incorporated inside of the CS/PVA fibers. Scaffolds were fully characterized via scanning electron microscopy, water contact angle, tensile measurement, hydration, protein adsorption, and wrinkled tests. Furthermore, viability of the buccal fat pad-derived mesenchymal stem cells (BFP-MSCs) was also investigated using MTT assay for up to 14 days while cultured on these scaffolds. Cell cycle assay was also performed to more detailed evaluation of the stem cells growth when grown on scaffolds (with and without Cur) compared with the culture plate. Results demonstrated that Cur loaded nanofibrous scaffold had more suitable capability for water absorption and mechanical properties compared with the scaffold without Cur and it could also support the stem cells viability and proliferation. Cur release profile showed a decreasing effect on BFP-MSCs viability in the initial stage, but it showed a positive effect on stem cell viability in a long-term manner. In general, the results indicated that this nanofibrous scaffold has great potential as a delivery of the Cur and BFP-MSCs simultaneously, and so holds the promising potential for use in various regenerative medicine applications.     

Adipose tissue-derived stem cells upon decellularized ovine small intestine submucosa for tissue regeneration: An optimization and comparison method

The extracellular matrix of different mammalian tissues is commonly used as scaffolds in the field of tissue engineering. One of these tissues, which has frequently been studied due to its structural and biological features, is the small intestine submucosal membrane. These research are mainly done on the porcine small intestine. However, a report has recently been published about a scaffold produced from the submucosal layer of the ovine small intestine. In the present study, ovine small intestine submucosal (OSIS) was decellularized in a modified manner and its histological, morphological, and biomechanical properties were studied. Decellularization was performed in two phases: physical and chemical. In this method, a chloroform-methanol mixture, enzymatic digestion, and a constant dose of sodium dodecyl sulfate (SDS) was used in the least agitation time and its histological property and biocompatibility were evaluated in the presence of adipose tissue-derived stem cells (ADSCs); furthermore, ADSCs were isolated with a simple method (modified physical washing non-enzymatic isolation). The results were showed that the use of OSIS could be effective and operative. Mechanical properties, histological structure and shape, and glycosaminoglycan content were preserved. In the SDS-treated group, more than 90% of the native cells of tissue were deleted, and also in this group, no toxicity was observed and cell proliferation was supported, compared to the untreated group. Therefore, our results indicate that ADSCs seeded on OSIS scaffold could be used as a new approach in regenerative medicine as hybrid or hydrogel application.     

A comparative study on nonviral genetic modifications in cord blood and bone marrow mesenchymal stem cells

The focus of both clinical and basic studies on stem cells is increasing due to their potentials in regenerative medicine and cell-based therapies. Recently stem cells have been genetically modified to enhance an existing character in or to bring a new property to them. However, accomplishment of declared goals requires detailed knowledge about their molecular characteristics which could be achieved by genetic modifications mostly through nonviral transfection strategies. Capable of differentiating into multiple cells, human unrestricted somatic stem cells (hUSSCs) and human mesenchymal stem cells (hMSCs) seem to be suitable candidates for transfection approaches. Involvement of microRNAs (miRNAs) in many biological processes makes their transfection evaluation valuable. Herein we investigated the efficacy and toxicity of four typically used transfection reagents (Arrest-In, Lipofectamine 2000, Oligofectamine and HiPerfect) systematically to deliver fluorescent labeled-miRNA and Green Fluorescent Protein (GFP) expressing plasmid into hUSSCs and hMSCs. The authenticity of stem cells was verified by differentiation experiments along with flow cytometry of surface markers. Our study revealed that stemness properties of these stem cells were not affected by transient transfection. Moreover the ratios of cell viability and transfection efficiency in both analyzed stem cells were reversed. Considering cell viability, the highest fraction of GFP-expressing cells was obtained using Oligofectamine (~50%) while the highest transfection rate of miRNA was achieved by Lipofectamine 2000 (~90%). Moreover dependency of hMSCs to size of transfected nucleic acid and time-dependency of Oligofectamine and their affection on the yield of transfection were observed. Cytotoxicity assessments also showed that hUSSCs are sensitive to HiPerFect. In addition cells treated by Lipofectamine showed morphological changes. Representing the efficient nucleic acid transfection, our research facilitates comprehensive genetic modification of stem cells and demonstrates powerful approaches to understand stem cell molecular regulation mechanisms, which eventually improves nonviral cell-mediated gene therapy.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-012-9430-9) contains supplementary material, which is available to authorized users.